Abstract
Ssn3, also known as Cdk8, is a member of the four protein Cdk8 submodule within the multi-subunit Mediator complex involved in the co-regulation of transcription. In Candida albicans, the loss of Ssn3 kinase activity affects multiple phenotypes including cellular morphology, metabolism, nutrient acquisition, immune cell interactions, and drug resistance. In these studies, we generated a strain in which Ssn3 was replaced with a functional variant of Ssn3 that can be rapidly and selectively inhibited by the ATP analog 3-MB-PP1. Consistent with ssn3 null mutant and kinase dead phenotypes, inhibition of Ssn3 kinase activity promoted hypha formation. Furthermore, the increased expression of hypha-specific genes was the strongest transcriptional signal upon inhibition of Ssn3 in transcriptomics analyses. Rapid inactivation of Ssn3 was used for phosphoproteomic studies performed to identify Ssn3 kinase substrates associated with filamentation potential. Both previously validated and novel Ssn3 targets were identified. Protein phosphorylation sites that were reduced specifically upon Ssn3 inhibition included two sites in Flo8 which is a transcription factor known to positively regulate C. albicans morphology. Mutation of the two Flo8 phosphosites (threonine 589 and serine 620) was sufficient to increase Flo8-HA levels and Flo8 dependent transcriptional and morphological changes, suggesting that Ssn3 kinase activity negatively regulates Flo8.Under embedded conditions, when ssn3Δ/Δ and efg1Δ/Δ mutants were hyperfilamentous, FLO8 was essential for hypha formation. Previous work has also shown that loss of Ssn3 activity leads to increased alkalinization of medium with amino acids. Here, we show that the ssn3Δ/Δ medium alkalinization phenotype, which is dependent on STP2, a transcription factor involved in amino acid utilization, also requires FLO8 and EFG1. Together, these data show that Ssn3 activity can modulate Flo8 and its direct and indirect interactions in different ways, and underscores the potential importance of considering Ssn3 function in the control of transcription factor activities.
Highlights
One of the important roles of the Mediator transcriptional co-regulatory complex is to link the activity of promoter-bound transcriptional factors to the basal transcription machinery
Author summary In Candida albicans, Ssn3 kinase activity co-regulates the transcription of numerous genes involved in hyphal growth, metabolism and nutrient acquisition, immune cell interactions, and drug resistance
We identified two phosphosites in Flo8, a regulator of morphology and virulence, that were shown to negatively regulate Flo8 levels and activity
Summary
One of the important roles of the Mediator transcriptional co-regulatory complex is to link the activity of promoter-bound transcriptional factors to the basal transcription machinery. The Cdk module of Mediator plays important roles in modulating the activity of Mediator itself as well as the activity of transcription factors, among other proteins. The catalytic component of the Cdk module, is a cyclin-dependent like kinase and its activity depends on the cyclin-like protein Ssn. The Cdk kinase has been shown to be important for regulation during metabolism and morphology [1,2]. The Cdk module is of particular interest for its role in transitions between growth conditions and during development when cells need to rapidly make coordinated changes to the activities of certain transcription factors at specific promoters as well as for its role in maintaining proper levels of constitutive promoter activation [11]
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